This renewal grant addresses questions about the mechanism and regulation of fibronectin assembly. Lysophosphatidic acid (LPA) rapidly up-regulates the ability of osteosarcoma cells or fibroblasts to bind fibronectin or its N-terminal 70-kDa fragment and induces cells to flatten, contract, and develop filopodia. Binding sites for fibronectin are in linear arrays on or near filopodia. The 70-kDa fragment is crosslinked by transglutaminase or a photoactivable bifunctional linker to large (apparent size of 3-mDa) trypsin-sensitive molecules that, as assessed by immunological reactivity, do not represent preassembled fibronectin. The aims of the renewal are to characterize the 3-mDa molecules, learn how stimulation of cells with LPA causes the 3-mDa molecules to express binding sites for assembling fibronectin, and learn the interactions responsible for formation of fibronectin multimers that are insoluble in sodium dodecyl sulfate (SDS). We will purify the 3-mDa molecules, do microsequencing, and produce antibodies. The antibodies should immunoprecipitate crosslinked 70-kDa fragment, colocalize with the fragment in linear arrays on cell surfaces, and block binding of fibronectin to cell surfaces. If the 3-mDa molecules are heretofore unknown, the sequencing data and/or antibodies will be used to clone them. We will test the hypothesis that LPA initiates a pathway involving activation of Rho, cytoskeletal rearrangement tethering of integrins, and integrin-dependent tension on the 3-mDa molecules. These experiments will utilize beta/1-integrin null cells cultured on vitronectin. Such cells must be transfected with beta/1 cDNA to be assembly competent and therefore offer the opportunity to learn the structural requirements for participation of beta/1-integrins in the assembly process. Finally, we will test if formation of SDS-insoluble fibronectin multimers involves a strong noncovalent interaction between a tryptophan in module I-4, which in protomeric fibronectin interacts with the neighboring I-5 module, and modules I-9/III-1 of an adjacent fibronectin subunit.